Non-receptor tyrosine kinases are intracellular enzymes which, in the presence of ATP phosphorylate proteins at tyrosine residues. These enzymes are key regulators of cellular signal transduction, leading to the activation, proliferation and differentiation of cells. The Src family of non-receptor tyrosine kinases comprises eight members: Src, Yes, Fyn, Lck, Lyn, Hck, Blk and Fgr, of which the first three kinases are ubiquitously expressed and the latter five kinases are primarily found in the haematopoietic system (Benatie et al. Current medical chemistry, 2008, 15, 1154-1165; Bogon et al. Oncogene 2004. 23, 7918-7927; Parsons et al. Oncogene, 2004. 23, 7906-7909). Members of the Src family display a conserved domain organization, which contains a myristoylated N-terminal domain, a unique region, a Src-homology 2 (SH2) domain, a SH3 domain, a tyrosine kinase domain and a C-terminal negative regulatory domain.
The Scr family members expressed in the haematopoietic system play an important role in the regulation of cells of the immune system and enhanced activity of these kinases has been implicated in a variety of malignant and non-malignant proliferative disorders. A particular Src family kinase of interest is the lymphocyte specific kinase (Lck) p56, which is primarily expressed in T lymphocytes and NK T cells. Lck, a proximal tyrosine kinase, is crucial for the initiation of signal transduction via the T cell receptor (TCR), which activates T lymphocytes. Upon antigen recognition, via MHC-TCR/peptide interaction, Lck is recruited to the TCR complex via the CD4/8 co-receptor, where it phosphorylates specific tyrosine residues in the immotyrosine-based activation motifs (ITAMs) located within the TCR ζchain. This phosphorylation event is crucial for the recruitment of the Syk-family kinase ZAP70 via SH2 interaction. Sequential phosphorylation of ZAP70 by Lck activates downstream signal transduction, leading to the activation and recruitment of other kinase family members and enzymes, resulting Ca2+ release leading towards full activation of the T cell (Palacios et al. Oncogene, 2004; 23, 7990-8000; Iwashima et al. 1994; 263, 1136-1139; Weiss A et al. 1994; 76, 263-274). Inhibition of Lck kinase activity will arrest TCR-mediated activation of ZAP70 and downstream mobilization of Ca2+ release, thereby inhibiting antigen-dependent activation of T lymphocytes.
Lck kinase inhibitors are useful for the treatment of chronic T cell disorders like multiple sclerosis and rheumatoid arthritis, as well as acute inflammatory disorders in which T cells play a prominent role including transplant rejection, atopic dermatitis and delayed type hypersensitivity (DTH). There clearly is a need for low molecular weight inhibitors of Lck for the treatment of chronic T cell disorders.
In WO2001019829 the use of pyrazolopyrimidine derivatives is directed to a method for the inhibition of, among others, Lck. The pyrazolopyrimidine derivatives of said patent application which is inserted by reference allow many different substituents as can be deduced from the definitions for substituents G, R2 and R3 in said pyrazolopyrimidine derivatives which are listed in WO2001019829. U.S. Pat. No. 7,459,554 describes imidazopyrazines tyrosine kinase inhibitors, including Lck. Also in this series of compounds, a very large variety of substituents is allowed as follows from the definitions for R1 and Q1 and their substituents as indicated in columns 10 to 15 of U.S. Pat. No. 7,459,554. Compounds according to WO2001019829 or U.S. Pat. No. 7,459,554 have an optionally substituted 8-amino substituent (NHR3 or NH2, respectively) (numbering according to Formula I). Furthermore, a large flexibility in the type and size of substituents is allowed.
Crystal structures of three Src family members: Src, Hck and Lck have enabled a detailed view of how the Src family of kinases is regulated, and the way in which small molecule inhibitors can inactivate these enzymes [Williams et al., JBC, 284, 284-291 (2009)]
Binding studies of Lck and ligands like 4-amino-1-cyclohexyl-3-phenyl-pyrazolo[3,4-d]pyrimidines reveal the 4-amino group (the 4-position in this compound is comparable with the 8-position in Formula I) making a key H-bond donor contact to the backbone C═O of Glu317 whilst the N5 pyrimidine nitrogen contacts the backbone NH of Met319 [Barbani et al., Bioorg. Med. Chem. Lett. 14, 2004 2613; Abbott et al., Bioorg. Med. Chem. Lett. 17, 1167-1171 (2007)]. All these studies reveal the presence of such an H-bond to the backbone C═O of Glu317. A similar binding has been mode has been observed for ATP-analogues and imidazo[1,5a]pyrazines (Strucuture 7(6) p651 (1999)) (EMBOj 27(14) 1985-1994 (2008)).
Binding studies of Lck and ligands like 4-amino-1-cyclohexyl-3-phenyl-pyrazolo[3,4-d]pyrimidines further reveal that the 3-phenyl group and its substituents (corresponding with R3 in Formula 1) extends into the hydrophobic pocket of Lck and that the 1-cyclohexyl group and its substituents (corresponding with R4 in Formula 1) extends into the solvent exposed region of the Lck binding pocket [Barbani et al., Bioorg. Med. Chem. Lett. 14, 2004 2613; Abbott et al., Bioorg. Med. Chem. Lett. 17, 1167-1171 (2007)].
We have found a series of compounds that lack the H-bond donor capacity to make an H-bond contact with the backbone C═O of Glu 317 and are surprisingly effective inhibitors of Lck.